Jingfang Granule Ameliorates Autoimmune Hepatitis Induced By Concanavalin A In Mice Via Regulating Tricarboxylic Acid Cycle

Objective:The purpose of this study is to study the protective effect of Jingfang Granule(JFG)on mice autoimmune hepatitis(AIH)through a concanavalin A(Con A)induced AIH model and further to explore the mechanism based on the proteomics and metabolomics techniques.Methods:Male ICR mice were randomly divided into 5 groups(n=10):Control group(Control),Model group(Model),low-dose of JFG group(JFGL),middle-dose of JFG group(JFG M),and high-dose of JFG group(JFG H).Except the Control group,the AIH model was established by intravenous injection(i.v.)of 10 mg/kg Con A.Mice in the JFG groups were administered with 1.0(JFG L),2.0(JFG M),and 4.0(JFG H)g/kg/d JFG via intragastric gavage(i.g.)1 h after the model were established respectively.The Mice in the Control and Model groups were administered the same volume of purified water.The mortality of mice was recorded.Then,peripheral blood and liver tissue samples were randomly collected after anesthesia with 1%pentobarbital solution 24 h after administration.The levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)were measured.The histopathological changes of liver were detected by hematoxylin and eosin(H&E)staining.Terminal deoxynucleotidyl transfer mediated(dUTP)biotin nick end labeling(TUNEL)kit was used to detect the expression of hepatocyte apoptosis.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of Interleukin(IL)-1β、IL-4、IL-6、IL-10、Tumor necrosis factor(TNF)-α、Interferon(IFN)γ in liver tissue.The level changes of liver tissue related proteins included BCL-2-associated X protein(Bax)、B-cell lymphoma-2(Bcl-2)、Cleaved caspase-3、Toll-like receptor 4(TLR4)、Myeloid differentiation factor 88(MyD88)、Tumor necrosis factor receptor associated factor(TRAF6)、Phosphorylation Nuclear factor kappa-B(p-NF-κB),Phosphorylated mitogen activated protein kinase p38 antibody(p-P38),phosphorylated extracellular signal regulated kinase 1/2(p-ERK1/2)and phosphorylated c-Jun N-terminal kinase(p-JNK)was used to detect Western blot.In order to further study the pharmacological mechanism of JFG in treating AIH,this study carried out proteomic analysis by label-free unlabeled quantitative technology and metabonomic analysis by liquid chromatography-mass spectrometry(LC-MS)technology to study the regulatory network of JFG in liver tissue,and the expression of related proteins ATP citrate lyase(ACLY)、Isocitrate dehydrogenase(IDH1)、Citrate synthase(CS)、Succinate-CoA ligase(SUCLG1)and Succinate-CoA ligase ADP-forming(SUCLA2)were detected by Western blot.Results:1.This study successfully established a Con A induced autoimmune hepatitis model,which showed a higher mortality rate in mice and a significant increase in the activity of serum ALT,AST,and LDH.JFG substantially alleviated Con A-induced hepatitis,which was demonstrated by increased survival rates of mice and lower activity of serum ALT,AST and LDH in JFG groups compared to the Model group.2.HE staining showed that the necrosis of liver cells and the degree of inflammatory infiltration in JFG group were significantly reduced compared with the Model group.3.TUNEL staining results indicated that a huge number of positive hepatocyte apoptosis were detected in the Model group,while the number of positive hepatocyte apoptosis was decreased significantly after JFG H treatment.The western blot results showed that the expression of Bax and cleaved caspase 3 in the Model group was evidently increased compared with the Control group,and the expression of Bcl-2 was decreased in the Model group.JFG H could reduce the expression of Bax and Cleaved caspase-3,and increased the expression of Bcl-2.4.The Nuclear factor kappa-B(NF-Kb)and Mitogen-activated protein kinase(MAPK)signal pathways were activated after Con A injection in mice.JFG H also regulated the production of multiple cytokines including IL-1β,IL-4,IL-6,TNF-α,IFN-γ and IL-10 by inhibiting NF-κB and MAPK pathways,and thus played an anti-inflammatory role.5.The results of proteomics and metabolomics showed that a total of 4359 differential proteins and 45 differential metabolic markers were detected.According to the integrated proteomics and metabolomics analysis of differential proteins and metabolites,it was found that the effect of JFG in treating AIH could be related to the regulation of tricarboxylic acid(TCA)cycle.6.Analyze differential protein expression in the TCA cycle using western blot.As shown in western blot,the levels of ACLY were significantly increased and the expression of IDH1,CS,SUCLG1 and SUCLA2 were significantly decreased in Model group compared with the Control group.Moreover,JFG H treatment reduced the expressions of ACLY and augmented the expression of IDH1,CS,SUCLG1 and SUCLA2 compared with the Model group.This study found that JFG H may regulate the production of inflammatory mediators by regulating the TCA cycle,thereby regulating the NF-κB and MAPK protein signaling pathways and exerting anti-inflammatory effects.Conclusion:This study found for the first time that JFG had a significant protective effect on Con A-induced AIH.Combined with proteomics and metabolomics technology,this protection effect is closely related to the regulative effect of JFG on TCA cycle to suppress the liver inflammation.This research further expands the clinical value of JFG,which provide a potential candidate drugs and theoretical basis for the application of JFG in the treatment of AIH.